Recombinant Immunotoxin Research Articles

Paclitaxel synergizes with exposure time adjusted CD22-targeting immunotoxins against B-cell malignancies.

CD22-targeted recombinant immunotoxins (rIT) are active in hairy cell leukemia or acute lymphoblastic leukemia (ALL), but not in mantle cell lymphoma (MCL) patients. The goal was to enhance rIT efficacy in vivo and to define a strong combination treatment. Activity of Moxetumomab pasudotox (Moxe) and LR combined with paclitaxel was tested against MCL cell lines in vitro and as bolus doses or continuous infusion in xenograft models. In the KOPN-8 ALL xenograft, Moxe or paclitaxel alone was active, but all mice died from leukemia; when combined, 60% of the mice achieved a sustained complete remission. Against MCL cells in vitro, LR was more active than Moxe and the cells had to be exposed to rIT for more than 24 hours for them to die. To maintain high blood levels in vivo, LR was administered continuously by 7-day pumps achieving a well-tolerated steady plasma concentration of 45 ng/ml. In JeKo-1 xenografts, continuously administered LR was 14-fold more active than bolus doses and the combination with paclitaxel additionally improved responses by 135-fold. Maintaining high rIT-plasma levels greatly improves responses in the JeKo-1 model and paclitaxel substantially enhances bolus and continuously infused rIT, supporting a clinical evaluation against B-cell malignancies.

HER2-specific recombinant immunotoxin 4D5scFv-PE40 passes through retrograde trafficking route and forces cells to enter apoptosis

Immunotoxin 4D5scFv-PE40 is a recombinant protein that comprises 4D5scFv antibody as a targeting module and fragment of Pseudomonas exotoxin A as an effector (toxic) one. The immunotoxin has shown pronounced antitumor effect on cancer cells overexpressing HER2 receptor in vitro and on HER2-positive experimental tumors in vivo. We clarified the mechanism of 4D5scFv-PE40 activity that is of particular importance in the case of targeted therapeutic agent aimed at personalizing treatment of disease in relation to molecular genetic characteristics of each patient. After specific binding to HER2 on the cell surface and clathrin-mediated endocytosis the immunotoxin passes through retrograde trafficking route. During this route the immunotoxin molecule is supposed to undergo enzymatic processing that ends in separation of C-terminal and N-terminal fragments of the immunotoxin. Finally, C-terminal functionally active fragment of 4D5scFv-PE40 arrests protein synthesis in cytoplasm followed by cell death via apoptosis.

Humanized CD7 nanobody-based immunotoxins exhibit promising anti-T-cell acute lymphoblastic leukemia potential.

BackgroundNanobodies, named as VHHs (variable domain of heavy chain of HCAb [heavy-chain antibodies]), are derived from heavy-chain-only antibodies that circulate in sera of camelids. Their exceptional physicochemical properties, possibility of humanization, and unique antigen recognition properties make them excellent candidates for targeted delivery of biologically active components, including immunotoxins. In our previous efforts, we have successfully generated the monovalent and bivalent CD7 nanobody-based immunotoxins, which can effectively trigger the apoptosis of CD7-positive malignant cells. To pursue the possibility of translating those immunotoxins into clinics, we humanized the nanobody sequences (designated as dhuVHH6) as well as further truncated the Pseudomonas exotoxin A (PE)-derived PE38 toxin to produce a more protease-resistant form, which is named as PE-LR, by deleting majority of PE domain II.Methods and resultsThree new types of immunotoxins, dhuVHH6-PE38, dVHH6-PE-LR, and dhuVHH6-PE-LR, were successfully constructed. These recombinant immunotoxins were expressed in Escherichia coli and showed that nanobody immunotoxins have the benefits of easy soluble expression in a prokaryotic expression system. Flow cytometry results revealed that all immunotoxins still maintained the ability to bind specifically to CD7-positive T lymphocyte strains without binding to CD7-negative control cells. Laser scanning confocal microscopy revealed that these proteins can be endocytosed into the cytoplasm after binding with CD7-positive cells and that this phenomenon was not observed in CD7-negative cells. WST-8 experiments showed that all immunotoxins retained the highly effective and specific growth inhibition activity in CD7-positive cell lines and primary T-cell acute lymphoblastic leukemia (T-ALL) cells. Further in vivo animal model experiments showed that humanized dhuVHH6-PE38 immunotoxin can tolerate higher doses and extend the survival of NOD-Prkdcem26Il2rgem26Nju (NCG) mice transplanted with CEM cells without any obvious decrease in body weight. Further studies on NCG mice model with patient-derived T-ALL cells, dhuVHH6-PE38 treatment, significantly prolonged mice survival with ~40% survival improvement. However, it was also noticed that although dhuVHH6-PE-LR showed strong antitumor effect in vitro, its in vivo antitumor efficacy was disappointing.ConclusionWe have successfully constructed a targeted CD7 molecule-modified nanobody (CD7 molecule-improved nanobody) immunotoxin dhuVHH6-PE38 and demonstrated its potential for treating CD7-positive malignant tumors, especially T-cell acute lymphoblastic leukemia.

Bioinformatic prediction and experimental validation of a PE38-based recombinant immunotoxin targeting the Fn14 receptor in cancer cells.

AFn14R can serve as an ideal target for cancer immunotherapy. Here, a combined bioinformatic and experimental approach was applied to characterize an immunotoxin consisting of single-chain variable fragment antibody that targets Fn14 and a toxin fragment (PE38). Flow cytometry results showed that the rate of PE38-P4A8 binding to Fn14 was approximately 60 and 40% in HT-29 and A549 cells, respectively. Moreover, 1 ng/µl of immunotoxin was able to lyse approximately 53 and 41% of HT-29 and A549, respectively. PE38-P4A8 showed stability in mouse serum (∼90%) after 3-h incubation. Most importantly, using bioinformatics for determining the structure and function of fusion proteins can be very helpful in designing of experiments. Coupled with bioinformatics, experimental approaches revealed that PE38-P4A8 could be used as a promising therapeutic agent for cancer cells expressing Fn14.

A combinatorial immunotherapy for malignant brain tumors: D2C7 immunotoxin and immune checkpoint inhibitors.

102 Background: Immunotoxins can induce direct and rapid cytotoxicity by targeting specific tumor antigens. D2C7 is a unique recombinant immunotoxin targeting EGFRwt/EGFRvIII, two frequently overexpressed proteins on gliomas, and is currently being tested in a phase I/II clinical trial (NCT02303678) for recurrent malignant gliomas. Immunotoxins have also been shown to induce a secondary antitumor immune response via stimulation of cytotoxic T lymphocytes (CTLs). Immune checkpoint inhibitors, which have successfully treated several advanced tumors by promoting the antitumor function of CTLs, may further enhance this immunotoxin-induced antitumor response. Thus, we hypothesize that combining D2C7 with immune checkpoint inhibitors will promote long-term tumor regression due to primary cytotoxicity and enhanced anti-tumor immunity. Methods: We developed a CT2A-mD2C7 mouse glioma cell line with robust in vitro cytotoxicity of D2C7 (IC50= 0.47 ng/mL). In vivo anti-tumor efficacy was evaluated by intratumoral injection of D2C7 combined with intraperitoneal injection of anti-CTLA4 or anti-PD1 antibodies in single-side and bilateral subcutaneous (SC) CT2A-mD2C7 glioma models in C57BL/6 immunocompetent mice. Results: In the single-side model, D2C7 monotherapy and combinatorial therapy showed a significant tumor growth delay (P < 0.01). Complete regression ( ≥ 40%) was only observed in combinatorial therapy groups. All cured mice rejected the rechallenging of CT2A parental cells in the contralateral flank and the subsequent rechallenging of CT2A-mD2C7 cells in the brain. In the bilateral model, the larger right tumors were treated with D2C7/anti-CTLA4/anti-PD1 monotherapy or D2C7+anti-CTLA4/PD1 combinatorial therapy, while the left tumors were untreated by D2C7. In the groups where the right tumors were treated with monotherapy or combinatorial therapy, the left untreated tumors also grew much slower. Furthermore, the combinatorial therapy led to the most significantly delayed growth of the left untreated tumors (P < 0.05). Conclusions: Immune checkpoint inhibitors can enhance D2C7-induced anti-tumor immunity to achieve a synergistic long-term tumor regression.

Anti-tumor effects of a recombinant anti-prostate specific membrane antigen immunotoxin against prostate cancer cells

BackgroundTo evaluate anti-prostate cancer effects of a chimeric tumor-targeted killer protein.MethodsWe established a novel fusion gene, immunocasp-3, composed of NH2-terminal leader sequence fused with an anti-prostate-specific membrane antigen (PSMA) antibody (J591), the furin cleavage sequences of diphtheria toxin (Fdt), and the reverse coding sequences of the large and small subunits of caspase-3 (revcaspase-3). The expressing level of the immunocasp-3 gene was evaluated by using the reverse transcription-PCR (RT-PCR) and western blot analysis. Cell viability assay and cytotoxicity assay were used to evaluate its anti-tumor effects in vitro. Apoptosis was confirmed by electron microscopy and Annexin V-FITC staining. The antitumor effects of immunocasp-3 were assessed in nude mice xenograft models containing PSMA-overexpressing LNCaP cells.ResultsThis study shows that the immunocasp-3 proteins selectively recognized and induced apoptotic death in PSMA-overexpressing LNCaP cells in vitro, where apoptotic cells were present in 15.3% of the cells transfected with the immunocasp-3 expression vector at 48 h after the transfection, in contrast to 5.5% in the control cells. Moreover, LNCaP cells were significantly killed under the condition of the co-culture of the immunocasp-3-secreting Jurkat cells and more than 50% of the LNCaP cells died when the two cell lines were co-cultured within 5 days. In addition, The expression of immunocasp-3 also significantly suppressed tumor growth and greatly prolonged the animal survival rate in vivo.ConclusionA novel fusion gene, immunocasp-3, may represent a viable approach to treating PSMA-positive prostate cancer.

Rational design of low immunogenic anti CD25 recombinant immunotoxin for T cell malignancies by elimination of T cell epitopes in PE38

LMB-2, is a potent recombinant immunotoxin (RIT) that is composed of scFv antibody that targets CD25 (Tac) and a toxin fragment (PE38). It is used to treat T cell leukemias and lymphomas. To make LMB-2 less immunogenic, we introduced a large deletion in domain II and six point mutations in domain III that were previously shown to reduce T cell activation in other RITs. We found that unlike other RITs, deletion of domain II from LMB-2 severely compromised its activity. Rather than deletion, we identified T cell epitopes in domain II and used alanine substitutions to identify point mutations that diminished those epitopes. The novel RIT, LMB-142 contains a 38kDa toxin and nine point mutations that diminished T cell response to the corresponding peptides by an average of 75%. LMB-142 has good cytotoxic activity and has lower nonspecific toxicity in mice. LMB-142 should be more efficient in cancer therapy because more treatment cycles can be given.

CD25 targeted therapy of chemotherapy resistant leukemic stem cells using DR5 specific TRAIL peptide

Chemotherapy resistant leukemic stem cells (LSCs) are being targeted as a modern therapeutic approach to prevent disease relapse. LSCs isolated from methotrexate resistant side population (SP) of leukemic cell lines HL60 and MOLT4 exhibited high levels of CD25 and TRAIL R2/DR5 which are potential targets. Recombinant immunotoxin conjugating IL2α with TRAIL peptide mimetic was constructed for DR5 receptor specific targeting of LSCs and were tested in total cell population and LSCs. IL2-TRAIL peptide induced apoptosis in drug resistant SP cells from cell lines and showed potent cytotoxicity in PBMCs derived from leukemic patients with an efficacy of 81.25% in AML and 100% in CML, ALL and CLL. IL2-TRAIL peptide showed cytotoxicity in relapsed patient samples and was more effective than TRAIL or IL2-TRAIL proteins. Additionally, DR5 specific IL2-TRAIL peptide was effective in targeting and killing LSCs purified from cell lines [IC50: 952nM in HL60, 714nM in MOLT4] and relapsed patient blood samples with higher efficacy (85%) than IL2-TRAIL protein (46%). Hence, CD25 and DR5 specific targeting by IL2-TRAIL peptide may be an effective strategy for targeting drug resistant leukemic cells and LSCs.

Recombinant Immunotoxin Therapy of Glioblastoma: Smart Design, Key Findings, and Specific Challenges.

Recombinant immunotoxins (RITs) refer to a group of recombinant protein-based therapeutics, which consists of two components: an antibody variable fragment or a specific ligand that allows RITs to bind specifically to target cells and an engineered toxin fragment that kills the target cells upon internalization. To date, over 1,000 RITs have been generated and significant success has been achieved in the therapy of hematological malignancies. However, the immunogenicity and off-target toxicities of RITs remain as significant barriers for their application to solid tumor therapy. A group of RITs have also been generated for the treatment of glioblastoma multiforme, and some have demonstrated evidence of tumor response and an acceptable profile of toxicity and safety in early clinical trials. Different from other solid tumors, how to efficiently deliver the RITs to intracranial tumors is more critical and needs to be solved urgently. In this article, we first review the design and expression of RITs, then summarize the key findings in the preclinical and clinical development of RIT therapy of glioblastoma multiforme, and lastly discuss the specific issues that still remain to forward RIT therapy to clinical practice.

Production and quality control assessment of a GLP-grade immunotoxin, D2C7-(scdsFv)-PE38KDEL, for a phase I/II clinical trial.

D2C7-(scdsFv)-PE38KDEL (D2C7-IT) is a novel recombinant Pseudomonas exotoxin A-based immunotoxin (IT), targeting both wild-type epidermal growth factor receptor (EGFRwt) and mutant EGFR variant III (EGFRvIII) proteins overexpressed in glioblastomas. Initial pre-clinical testing demonstrated the anti-tumor efficacy of D2C7-IT against orthotopic glioblastoma xenograft models expressing EGFRwt, EGFRvIII, or both EGFRwt and EGFRvIII. A good laboratory practice (GLP) manufacturing process was developed to produce sufficient material for a phase I/II clinical trial. D2C7-IT was expressed under the control of the T7 promoter in Escherichia coli BLR (λ DE3). D2C7-IT was produced by a 10-L batch fermentation process and was then purified from inclusion bodies using anion exchange, size exclusion, and an endotoxin removal process that achieved a yield of over 300mg of purified protein. The final vialed batch of D2C7-IT for clinical testing was at a concentration of 0.12±0.1mg/mL, the pH was at 7.4±0.4, and endotoxin levels were below the detection limit of 10EU/mL (1.26EU/mL). The stability of the vialed D2C7-IT has been monitored over a period of 42months through protein concentration, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing, size exclusion chromatography, cytotoxicity, sterility, and pH measurements. The vialed D2C7-IT is currently being tested in a phase I/II clinical trial by intratumoral convection-enhanced delivery for 72h in patients with recurrent glioblastoma (NCT02303678, D2C7 for Adult Patients with Recurrent Malignant Glioma; clinicaltrials.gov ).

Mesothelin-targeted immunotoxin RG7787 has synergistic anti-tumor activity when combined with taxanes.

Recombinant immunotoxins (RITs) are antibody-based therapeutics that carry a toxin payload. The RG7787 RIT targets the cancer antigen mesothelin to deliver a recombinantly-engineered, reduced immunogenicity variant of Pseudomonas exotoxin A (PE) to the cytosol where it inhibits protein synthesis. Here we demonstrate that maximal doses of RG7787 temporarily halt growth of pancreatic cancer tumor xenografts, similar to the approved drugs gemcitabine and nab-paclitaxel, however, combination of the RIT with nab-paclitaxel produces durable complete regressions in most mice. Synergy between taxane and anti-MSLN RITs has been previously demonstrated in mouse models, but direct interaction of the combination in cell culture was not observed. Here, we show that this favorable interaction occurs in cell culture, is dependent on the dose and duration of RG7787 exposure, requires the catalytically active PE, and still occurs with RIT targeting a non-MSLN surface antigen. Unexpectedly, the combination does not increase RG7787-mediated protein synthesis inhibition nor perturb downstream apoptotic markers of RIT-mediated killing, but does augment levels of acetylated tubulin, a marker of taxane activity. Taken together, these data suggest that PE increases cell sensitivity to taxane-mediated killing by increasing taxane-mediated microtubule stability and priming cells for apoptosis by decreasing levels of the pro-survival factor Mcl-1.

5-Azacytidine Prevents Relapse from Discrete Bone Marrow Sites in Leukemia Xenografts Treated with CD22-Targeting Immunotoxin Moxetumomab Pasudotox

Background: Moxetumomab pasudotox (Moxe) is a recombinant immunotoxin that targets CD22. Moxe showed high clinical activity in a phase II trial against hairy cell leukemia (Kreitman et al, 2012) and a phase III trial has recently completed enrollment. Moxe also demonstrated objective responses in 30% of children with multiply relapsed acute lymphoblastic leukemia (ALL) (Wayne et al AACR 2014). In a xenograft model with the ALL cell line KOPN-8, Moxe as monotherapy clears the murine bone marrow (BM) from leukemia blasts (Mueller et al, CCR 2016). However, mice eventually relapse and die of progressive disease.Goal: To characterize the ALL cells responsible for relapse and to devise a relapse-preventing treatment.Methods/Results: The ALL cell lines KOPN-8 and REH were transduced with luciferase. The systemic bioluminescence signal in KOPN-8 bearing mice was abrogated after three i.v. bolus-doses of Moxe. Relapse was subsequently detected at various, apparently randomly distributed BM sites which possibly were the origin of systemic relapse. The systemic relapse remained Moxe-sensitive for several treatment cycles, but the bioluminescence signal at the discrete BM sites persisted. After several treatment cycles a Moxe-resistant systemic relapse emerged and mice died of progressive disease as determined by bioluminescence and flow cytometry. The Moxe-resistant cells showed a loss of CD10, and a 2.5-fold decrease in surface CD19 and CD22. CD22 mRNA levels and total CD22 protein by Western Blot were reduced up to 7-fold, indicating a profound reduction of intracellular CD22 in resistant cells. RNA deep sequencing revealed a global transcriptional change including down-regulation of B-cell specific genes suggesting a less mature state of KOPN-8 in the Moxe resistant blasts.We had previsouly demonstrated an epigenetic mechanism of Moxe resistance in vitro (Hu et al, Leuk Res 2013). Because most of the genetically homogeneous KOPN-8 cells growing in murine BM were Moxe sensitive but cells at discrete sites persisted, we hypothesized that locally induced epigenetic changes might partially explain the resistance. We therefore combined Moxe with the DNA-methyltransferase inhibitor 5-Azacytidine (5-AZA). KOPN-8 bearing mice treated with Moxe or 5-AZA alone survived longer than untreated mice, but eventually died from leukemia. However, mice that received the combination of 5-AZA and Moxe were disease free at 4 months after treatment by flow cytometry and histopathology. The combination of 5-AZA and Moxe also improved responses over each agent alone in a second ALL xenograft model with the ALL cell line REH.Conclusion: The relapse of KOPN-8 after treatment with Moxe, possibly from discrete BM sites is consistent with BM-niche induced resistance. This niche-induced resistance is further supported by locally persisting cells after repeated treatment cycles, while blasts that repopulate other BM sites remain rIT-sensitive. That relapse at discrete BM-sites is prevented by 5-AZA suggests that epigenetic modifications contribute to induction of localized resistance in vivo. rIT-resistant KOPN-8 relapse shows a profound reduction in total CD22 which may contribute to resistance to CD22-directed rIT. The marked increase of Moxeefficacy in vivo by the addition of 5-AZA makes this combination a promising approach for further clinical testing in children and adults with ALL. DisclosuresWayne:Kite Pharma: Honoraria, Other: Travel support, Research Funding; Spectrum Pharmaceuticals: Honoraria, Other: Travel Support, Research Funding; Pfizer: Consultancy, Honoraria, Other: Travel Support; Medimmune: Honoraria, Other: Travel Support, Research Funding; NIH: Patents & Royalties. Pastan:NIH: Patents & Royalties: Co-Inventor on NIH Patent.

Development and investigation of recombinant immunotoxin protein 4D5scFv-mCherry-PE(40).

Development of agents for theranostics implies combining the targeting module, the effector module, and the detection module within the same complex or recombinant protein. We have constructed, isolated, and characterized the 4D5scFv-mCherry-PE(40) protein, which exhibits fluorescent properties and specifically binds to cancer cells expressing the HER2 receptor and reduces their viability. The ability of the obtained targeted antitumor agent 4D5scFv-mCherry-PE(40) to selectively stain the HER2-positive cells and its highly selective cytotoxicity against these cells make the obtained targeted recombinant protein 4D5scFv-mCherry-PE(40) a promising theranostic agent for the diagnostics and therapy of HER2-positive human tumors.

Camelid VH H affinity ligands enable separation of closely related biopharmaceuticals.

Interest in new and diverse classes of molecules such as recombinant toxins, enzymes, and blood factors continues to grow for use a biotherapeutics. Compared to monoclonal antibodies, these novel drugs typically lack a commercially available affinity chromatography option, which leads to greater process complexity, longer development timelines, and poor platformability. To date, for both monoclonal antibodies and novel molecules, affinity chromatography has been mostly reserved for separation of process‐related impurities such as host cell proteins and DNA. Reports of affinity purification of closely related product variants and modified forms are much rarer. In this work we describe custom affinity chromatography development using camelid VHH antibody fragments as "tunable" immunoaffinity ligands for separation of product‐related impurities. One example demonstrates high selectivity for a recombinant immunotoxin where no binding was observed for an undesired deamidated species. Also discussed is affinity purification of a coagulation factor through specific recognition of the gamma‐carboxylglutamic acid domain.

Novel Antibody Therapeutics Targeting Mesothelin In Solid Tumors.

: BackgroundMonoclonal antibodies have become attractive clinical anti-cancer drugs in the last 3 decades due to their targeting specificity and suitable pharmacokinetic properties. Mesothelin is a tumor-associated antigen with limited expression in normal tissues. It is frequently over-expressed on the cell membrane of a number of epithelial malignancies (e.g. mesothelioma, pancreatic, ovarian, lung, triple negative breast and gastric cancers).MethodsMesothelin is validated as a suitable antibody target for cancer therapy. A number of novel antibody therapeutics targeting mesothelin in development are compared and their mechanisms of action are also discussed. Both basic science and clinical data are provided to give a complete veiw of how an agent is developed from bench to bedside.ResultsNovel antibody therapeutics, including unconjugated monoclonal antibodies, recombinant immunotoxins and antibody-drug conjugates, targeting mesothelin exert anti-tumor activities by different mechanisms of action. Based on the convincing preclinical data generated with these molecules, the antibody therapeutics have been brought into early clinical evaluation where initial promising results were obtained.ConclusionThese antibody therapeutics directed against mesothelin are expected to have different safety profiles, based on their different mechanism of action. Further clinical development will reveal which of these molecules shows the best efficacy and widest therapeutic window and thus is best suited to bring benefit to the patients.

Wide Variability in the Time Required for Immunotoxins to Kill B Lineage Acute Lymphoblastic Leukemia Cells: Implications for Trial Design.

Recombinant immunotoxins (rITs) targeting CD22 are highly active in hairy cell leukemia, but less so in acute lymphoblastic leukemia (ALL). This study aims to understand the variable activity of an rIT against ALL toward improving responses in clinical application. We determined in vitro activity of rITs by WST-8 assays and the time needed to kill ALL cell lines and patient-derived ALL blasts by flow cytometry. The findings were translated into two systemic ALL xenograft models. Differences in time needed to kill KOPN-8 cells for distinct rITs were addressed biochemically. In vitro activity (IC50) of anti-CD22 rIT varied 210-fold from 0.02 to 4.6 ng/mL. Activity also varied greatly depending on the time ALL cells were exposed to immunotoxin from < 30 minutes to > 4 days. For KOPN-8, the difference in exposure time was related to intracellular rIT processing. We showed in newly developed ALL xenograft models, where immunotoxins have a short half-life, that the needed exposure time in vitro predicted the responses in vivo By replacing bolus dose with small doses at frequent intervals or with continuous infusion, responses were substantially improved. We confirmed exposure time variability on patient-derived ALL samples and showed a correlation between exposure time needed to reach maximal cytotoxicity in vitro and their clinical response. The exposure time needed for rITs targeting CD22 to kill ALL cells varies widely. Our results suggest that ALL patients would have a better response rate to anti-CD22 immunotoxins if treated by continuous infusion rather than by bolus injections. Clin Cancer Res; 22(19); 4913-22. ©2016 AACR.

Application of bee venom and its main constituent melittin for cancer treatment.

Bee venom and its main constituent melittin (MEL) have been extensively studied in the treatment of tumors. However, the non-specific cytotoxicity and hemolytic activity have hampered the clinical application. Currently, a number of research groups have reported a series of optimization strategies, including gene therapy, recombinant immunotoxin incorporating MEL or MEL nanoparticles, targeting tumor cells to attenuate the cytotoxicity and improve its antitumor efficiency and therapeutic capabilities, which have shown very promising in overcoming some of these obstacles. In this review, we summarize the current knowledge regarding anticancer effects of bee venom and its main compound MEL on different kinds of tumor cells as well as elucidate their possible anticancer mechanisms. It could be concluded that MEL exerts multiple effects on cellular functions of cancerous cells such as proliferation, apoptosis, metastasis, angiogenesis as well as cell cycle, and the anticancer processes involve diverse signal molecules and regulatory pathways. We also highlight the recent research progress for efficient delivery of MEL peptide, thus providing new ideas and hopeful strategies for the in vivo application of MEL.

Humanization of high-affinity antibodies targeting glypican-3 in hepatocellular carcinoma.

Glypican-3 (GPC3) is a cell-surface heparan sulfate proteoglycan highly expressed in hepatocellular carcinoma (HCC). We have generated a group of high-affinity mouse monoclonal antibodies targeting GPC3. Here, we report the humanization and testing of these antibodies for clinical development. We compared the affinity and cytotoxicity of recombinant immunotoxins containing mouse single-chain variable regions fused with a Pseudomonas toxin. To humanize the mouse Fvs, we grafted the combined KABAT/IMGT complementarity determining regions (CDR) into a human IgG germline framework. Interestingly, we found that the proline at position 41, a non-CDR residue in heavy chain variable regions (VH), is important for humanization of mouse antibodies. We also showed that two humanized anti-GPC3 antibodies (hYP7 and hYP9.1b) in the IgG format induced antibody-dependent cell-mediated cytotoxicity and complement-dependent-cytotoxicity in GPC3-positive cancer cells. The hYP7 antibody was tested and showed inhibition of HCC xenograft tumor growth in nude mice. This study successfully humanizes and validates high affinity anti-GPC3 antibodies and sets a foundation for future development of these antibodies in various clinical formats in the treatment of liver cancer.

Glypican-3 Targeting Immunotoxins for the Treatment of Liver Cancer.

Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer, yet no effective therapeutics exist. This review provides an overview of the recent development of recombinant immunotoxins for the treatment of glypican-3 (GPC3) expressing HCC. GPC3 is a cell surface heparan sulfate proteoglycan that is overexpressed in HCC, but is absent from normal adult human tissues. Treatment of HCC with anti-GPC3 immunotoxins represents a new therapeutic option. Using phage display and hybridoma technologies, three high affinity antibodies (HN3, HS20 and YP7) have been generated against GPC3. Two of these antibodies (HN3 and HS20) have demonstrated the ability to inhibit Wnt/Yap signaling, leading to a reduction in liver cancer cell proliferation. By combining the HN3 antibody capable of inhibiting Wnt/Yap signaling with the protein synthesis inhibitory domain of the Pseudomonas exotoxin, a recombinant immunotoxin that exhibits a dual inhibitory mechanism was generated. This immunotoxin was found to be highly effective in the treatment of human HCCs in mouse xenograft models. Engineering of the toxin fragment to reduce the level of immunogenicity is currently being explored. The development of immunotoxins provides opportunities for novel liver cancer therapies.

Abstract 743: Tyrosine kinase discoidin domain receptor-1 (DDR1) regulates cytotoxicity of recombinant immunotoxin for cancer therapy

Abstract Discoidin domain receptor 1 (DDR1) is a collagen-activated tyrosine kinase that facilitates cell adhesion, migration, proliferation and matrix remodeling. The collagen/DDR1 axis is also thought to modulate tumor-stromal interaction and potentially affects tumor response to therapy. Mesothelin is a cell-surface protein over-expressed in several human cancers including mesothelioma, ovarian, lung, breast, and pancreatic cancers with limited expression on normal cells. RG7787 is a clinically optimized recombinant immunotoxin (RIT) consisting of a humanized anti-mesothelin Fab fused to domain III of Pseudomonas exotoxin A in which immunogenic B cell epitopes are silenced. To enhance therapeutic efficacy of RITs we conducted a kinome RNAi sensitization screen which identified DDR1 as a potential target. We hypothesized that DDR1 regulates RIT activity and its inhibition might enhance cell killing by RG7787. Knockdown of DDR1 by siRNA or treatment with inhibitor, ‘7rh’, greatly enhanced the cytotoxic activity of RG7787 in several cancer cell lines. Investigation into the mechanism of action showed DDR1 silencing was associated with a decrease in several ribosomal proteins and enhanced inhibition of protein synthesis. Induction of DDR1 expression or stimulation of DDR1 activity by collagen protected cancer cells from RG7787 killing, while DDR1 inhibition enhanced killing by RG7787. Moreover, the combination of immunotoxin and DDR1 inhibitor caused significantly more shrinkage of epithelial A431/H9 and pancreatic KLM1 tumor xenografts than either agent alone. These data demonstrate that DDR1 has a critical role in modulating immunotoxin activity. Inhibition of DDR1 represents a novel strategy to enhance therapeutic efficacy of RITs targeting mesothelin-expressing cancers. Citation Format: Fatima Ali-Rahmani, David Fitzgerald, Scott Martin, Paresma Patel, Marco Prunotto, Pinar Ormanoglu, Craig Thomas, Ira Pastan. Tyrosine kinase discoidin domain receptor-1 (DDR1) regulates cytotoxicity of recombinant immunotoxin for cancer therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 743.